Electrophoresis is a commonly used method for analysis, wherein charged molecules and particles migrate in a separation medium, usually a gel, which is subjected to an electrical field between two electrodes. Separation of proteins may be by isoelectric point (pI), molecular weight, electric charge, or a combination of these factors.
The separation gel is usually placed on a support and two opposing ends of the gel are contacted with an electrode buffer in solution or rigid form. The electrodes may be inserted in vessels containing the electrode buffers. The buffer solutions form both the electrolytic medium and a reservoir for ions to keep the pH and other parameters constant. After separation, the molecules are detected and identified in different manners: e.g. visually by staining the gel or by optical means such as scanning or imaging the stained gel or labeller samples by a laser scanner or the like.
Gel electrophoresis is today routinely used for separating biomolecules such as proteins, peptides, nucleic acids etc. Samples are handled in different types of screening, identifying (cell signaling, expression & purification) or in clinical tests. Protein samples can derivate from e.g. human, mammalian tissue, cell lysates or bacterial, insect or yeast cellular systems. The electrophoretic conditions for different types of molecules are different and have to be adapted in many cases. Thus, both the gel and the buffer solutions must often be chosen for each type of sample.
Labeling proteins with fluorescent dyes has become the method of choice for tracking and quantifying proteins. Fluorescent labeling results in good sensitivity and a broad linear detection range. It also presents a convenient alternative to protein staining methods and is a safer option to radioactive labeling.
The choice of dye and labeling conditions depend on the application. For immunological applications, e.g. antibody labeling, it is important to get high signal intensity and the dye-to-protein ratio is optimized accordingly. For electrophoresis it is also necessary to use a suitable dye-to-protein ratio, in this case to get both high signal intensity and sharp electrophoresis bands. Furthermore, for isoelectric focusing (IEF) electrophoresis it is necessary to use charge-matched dyes to not change the isoelectric point of the protein. Pre-labeling for electrophoresis is well known (see e.g. “Electrophoresis” by Anthony T. Andrews, Clarendon Press, Oxford, 1986).